Succinic acid, the first large quantity commodity chemical, which can be made by fermentation of plant biomass substrates, is presently produced from petrochemical feedstocks. Succinic acid is an intermediate of the citric acid cycle, and many anaerobic bacteria form succinate as a fermentation product. The bacterium Anaerobiospirillum succiniciproducens is able to produce 35-50 g/l of succinate, but Actinobacillus sp. produces 130Z 65-80g/l. Phosphoenolpyruvate (PEP) carboxykinase is the key, regulated enzyme condensing PEP and CO2 to form oxaloacetate, in the pathway leading to succinate. We have successfully cloned and expressed A. succiniciproducens PEP carboxykinase in E. coli. This 60.9 kDa monomeric recombinant enzyme, containing an N-terminal His tag, was purified and used for the development of an MS-based protein sequencing method. At present we have purified the Actinobacillus sp 130Z PEP carboxykinase. TMPP-Ac charge derivatization and peptide sequencing by MALDI-PSD-MS is being used to assist in cloning the corresponding gene.